Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions

Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in many organisms, including humans, and offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death. In this work, we studied the biophysical and microbiological characteristics of designed CAPs varying in hydrophobicity levels and charge distributions by a variety of biophysical and biochemical approaches, including in-tandem atomic force microscopy, attenuated total reflection-FTIR, CD spectroscopy, and SDS-PAGE. Peptide structural properties were correlated with their membrane-disruptive abilities and antimicrobial activities. In bacterial lipid model membranes, a time-dependent increase in aggregated β-strand-type structure in CAPs with relatively high hydrophobicity (such as KKKKKKALFALWLAFLA-NH(2)) was essentially absent in CAPs with lower hydrophobicity (such as KKKKKKAAFAAWAAFAA-NH(2)). Redistribution of positive charges by placing three Lys residues at both termini while maintaining identical sequences minimized self-aggregation above the dimer level. Peptides containing four Leu residues were destructive to mammalian model membranes, whereas those with corresponding Ala residues were not. This finding was mirrored in hemolysis studies in human erythrocytes, where Ala-only peptides displayed virtually no hemolysis up to 320 μM, but the four-Leu peptides induced 40-80% hemolysis at the same concentration range. All peptides studied displayed strong antimicrobial activity against Pseudomonas aeruginosa (minimum inhibitory concentrations of 4-32 μM). The overall findings suggest optimum routes to balancing peptide hydrophobicity and charge distribution that allow efficient penetration and disruption of the bacterial membranes without damage to mammalian (host) membranes.     

Rab5 proteins regulate activation and localization of target of rapamycin complex 1

The mechanistic target of rapamycin (mTOR) complex 1 is regulated by small GTPase activators and localization signals. We examine here the role of the small GTPase Rab5 in the localization and activation of TORC1 in yeast and mammalian cells. Rab5 mutants disrupt mTORC1 activation and localization in mammalian cells, whereas disruption of the Rab5 homolog in yeast, Vps21, leads to decreased TORC1 function. Additionally, regulation of PI(3)P synthesis by Rab5 and Vps21 is essential for TORC1 function in both contexts.     

A monoclonal antibody specific for Candida albicans Als4 demonstrates overlapping localization of Als family proteins on the fungal cell surface and highlights differences between Als localization in vitro and in vivo

The Candida albicans agglutinin-like sequence (ALS) family encodes large cell surface glycoproteins that function in adhesion of the fungus to host and abiotic surfaces. Monoclonal antibodies (mAbs) specific for each Als protein were developed to study Als localization on the C. albicans surface. An anti-Als4 mAb demonstrated that Als4 covers the surface of yeast cells, with a greater abundance of Als4 on cells grown at 30 °C compared to 37 °C. On germ tubes, Als4 is localized in a restricted area proximal to the mother yeast. Immunolabeling with several anti-Als mAbs showed overlapping localization of Als1 and Als4 on yeast cells and Als1, Als3 and Als4 on germ tubes. Overlapping localization of Als proteins was also observed on yeast and hyphae recovered from mouse models of disseminated and oral candidiasis. Differences between Als localization in vivo and in vitro suggested changes in regulation of Als production in the host compared to the culture flask. Characterization with the anti-Als mAbs reveals the simultaneous presence and differences in relative abundance of Als proteins, creating an accurate image of Als representation and localization that can be used to guide conclusions regarding individual and collective Als protein function.     

Synthesis and antiviral activity of novel HCV NS3 protease inhibitors with P4 capping groups

We have synthesized and evaluated a series of novel HCV NS3 protease inhibitors with various P4 capping groups, which include urea, carbamate, methoxy-carboxamide, cyclic carbamate and amide, pyruvic amide, oxamate, oxalamide and cyanoguanidine. Most of these compounds are remarkably potent, exhibiting single-digit to sub-nanomolar activity in the enzyme assay and cell-based replicon assay. Selected compounds were also evaluated in the protease-inhibitor-resistant mutant transient replicon assay, and they were found to show quite different potency profiles against a panel of HCV protease-inhibitor-resistant mutants.     

Dysfunction of organic anion transporting polypeptide 1a1 alters intestinal bacteria and bile acid metabolism in mice

Organic anion transporting polypeptide 1a1 (Oatp1a1) is predominantly expressed in liver and is able to transport bile acids (BAs) in vitro. Male Oatp1a1-null mice have increased concentrations of taurodeoxycholic acid (TDCA), a secondary BA generated by intestinal bacteria, in both serum and livers. Therefore, in the present study, BA concentrations and intestinal bacteria in wild-type (WT) and Oatp1a1-null mice were quantified to investigate whether the increase of secondary BAs in Oatp1a1-null mice is due to alterations in intestinal bacteria. The data demonstrate that Oatp1a1-null mice : (1) have similar bile flow and BA concentrations in bile as WT mice; (2) have a markedly different BA composition in the intestinal contents, with a decrease in conjugated BAs and an increase in unconjugated BAs; (3) have BAs in the feces that are more deconjugated, desulfated, 7-dehydroxylated, 3-epimerized, and oxidized, but less 7-epimerized; (4) have 10-fold more bacteria in the small intestine, and 2-fold more bacteria in the large intestine which is majorly due to a 200% increase in Bacteroides and a 30% reduction in Firmicutes; and (5) have a different urinary excretion of bacteria-related metabolites than WT mice. In conclusion, the present study for the first time established that lack of a liver transporter (Oatp1a1) markedly alters the intestinal environment in mice, namely the bacteria composition.     

An Outbreak of Tularemia in a Colony of Outdoor-Housed Rhesus Macaques (Macaca mulatta)

Since an epizootic and detection of clinical cases of tularemia (Francisella tularensis) in 1996 at the Oregon National Primate Research Center, only 8 cases were identified in the succeeding 13 y. However, within a period of 7 mo, primarily during Winter 2010, 6 rhesus macaques were confirmed positive for Francisella tularensis type B by the Centers for Disease Control and Prevention by culture and fluorescent antibody testing. All cases had similar gross pathologic findings, which included necrotizing splenitis and lymphadenitis. Recent colony management efforts have focused on minimizing nonhuman primate exposure to commonly observed reservoir species and controlling rodent access to corral-style housing. Strategies continue to evolve with regard to managing a large breeding colony of nonhuman primates in the presence of this threat.Abbreviation: ONPRC, Oregon National Primate Research CenterFrancisella tularensis, the causative agent of tularemia, is a small pleomorphic gram-negative coccobacillus.¹¹ Severe disease and potentially death in humans can result from exposure to as few as 10 cfu of this highly infectious organism.^7,10 The disease is also known as rabbit fever and deer-fly fever, reflecting 2 common sources of infection for humans.³ F. tularensis is classified by the United States Department of Health and Human Services as a Category A Select Agent.⁶ It is considered a potential agent of biologic warfare, and in fact, has been weaponized and stockpiled in the past.¹⁰ The 2 biovars that are referenced most frequently in published human and nonhuman primate literature are tularensis (type A) and holarctica (formerly paleartica; type B).^4,12,13,23 An additional biovar, novicida (type C), has been described, but its virulence in humans is decreased due to its lack of a capsule.¹⁰Tularemia is endemic to many parts of the northern hemisphere, which includes the region surrounding the Oregon National Primate Research Center (ONPRC), an AAALAC-accredited facility.²⁰ Tularemia has one of the broadest host ranges of all bacteria, encompassing well over 200 mammalian species primarily, in addition to birds, amphibians, fish, and various arthropods such as fleas, ticks, mosquitoes, and flies.^10,15,19,20 The ONPRC is located in a mixed forest and field environment which is bordered by wetlands and residential neighborhoods outside of Portland. More than 4500 nonhuman primates are housed here, and most live outdoors in breeding groups. Therefore, exposure to this potentially life-threatening and zoonotic pathogen is inevitable, due to its persistence in the environment and the close proximity of several reservoir species. Presumed reservoir species commonly observed at ONPRC include meadow voles (Microtus pennsylvanicus), brown rats (Rattus norvegicus), deer mice (genus Peromyscus), house mice (Mus musculus), and California ground squirrels (Spermophilus beecheyi). Potential arthropod vectors that are monitored regularly at ONPRC include biting flies and mosquitoes. At this time, testing of prospective rodent carriers for tularemia is ongoing; therefore, in the interest of caution, all of the rodent and arthropod species listed are considered potential carriers of the disease.Tularemia was first recognized at the ONPRC in 1996 during an epizootic that resulted in 24 deaths among corral-housed rhesus macaques. Serology results from banked sera and sera collected during and after the outbreak demonstrated a seroconversion rate of approximately 25% in 723 animals. During the succeeding 13 y, only 8 sporadic cases were diagnosed. However, within a period of 3 mo during the winter of 2010, 5 rhesus macaques were diagnosed with Francisella tularensis type B by the Centers for Disease Control and Prevention (Atlanta, GA). Four months later, an additional case was confirmed. All 6 macaques were younger than 1 y and were assigned to a breeding colony protocol approved by the ONPRC Animal Care and Use Committee. The current report describes the clinical signs and gross and histologic findings associated with these cases, as well as methods for prevention and control of future cases of disease.     

Differences in the curing of [PSI+] prion by various methods of Hsp104 inactivation

[PSI(+)] yeast, containing the misfolded amyloid conformation of Sup35 prion, is cured by inactivation of Hsp104. There has been controversy as to whether inactivation of Hsp104 by guanidine treatment or by overexpression of the dominant negative Hsp104 mutant, Hsp104-2KT, cures [PSI(+)] by the same mechanism- inhibition of the severing of the prion seeds. Using live cell imaging of Sup35-GFP, overexpression of Hsp104-2KT caused the foci to increase in size, then decrease in number, and finally disappear when the cells were cured, similar to that observed in cells cured by depletion of Hsp104. In contrast, guanidine initially caused an increase in foci size but then the foci disappeared before the cells were cured. By starving the yeast to make the foci visible in cells grown with guanidine, the number of cells with foci was found to correlate exactly with the number of [PSI(+)] cells, regardless of the curing method. Therefore, the fluorescent foci are the prion seeds required for maintenance of [PSI(+)] and inactivation of Hsp104 cures [PSI(+)] by preventing severing of the prion seeds. During curing with guanidine, the reduction in seed size is an Hsp104-dependent effect that cannot be explained by limited severing of the seeds. Instead, in the presence of guanidine, Hsp104 retains an activity that trims or reduces the size of the prion seeds by releasing Sup35 molecules that are unable to form new prion seeds. This Hsp104 activity may also occur in propagating yeast.     

Barriers to partnership working in public health: a qualitative study

Background

Public health provision in England is undergoing dramatic changes. Currently established partnerships are thus likely to be significantly disrupted by the radical reforms outlined in the Public Health White Paper. We therefore explored the process of partnership working in public health, in order to better understand the potential opportunities and threats associated with the proposed changes.

Methodology/Principal Findings

70 participants took part in an in-depth qualitative study involving 40 semi-structured interviews and three focus group discussions. Participants were senior and middle grade public health decision makers working in Primary Care Trusts, Local Authorities, Department of Health, academia, General Practice and Hospital Trusts and the third sector in England. Despite mature arrangements for partnership working in many areas, and much support for joint working in principle, many important barriers exist. These include cultural issues such as a lack of shared values and language, the inherent complexity of intersectoral collaboration for public health, and macro issues including political and resource constraints. There is particular uncertainty and anxiety about the future of joint working relating to the availability and distribution of scarce and diminishing financial resources. There is also the concern that existing effective collaborative networks may be completely disrupted as the proposed changes unfold. The extent to which the proposed reforms might mitigate or potentiate these issues remains unclear. However the threats currently remain more salient than opportunities.

Conclusions

The current re-organisation of public health offers real opportunity to address some of the barriers to partnership working identified in this study. However, significant threats exist. These include the breakup of established networks, and the risk of cost cutting on effective public health interventions.     

Mutations in Escherichia coli aceE and ribB Genes Allow Survival of Strains Defective in the First Step of the Isoprenoid Biosynthesis Pathway

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.